Experimental ‘hindbrain related’ syringomyelia: some mechanisms of spinal cord damage

Numerous experimental observation and clinical researches demonstrated that the majority of the forms of syringomyelia are found in combination with inherent or acquired hindbrain abnormalities [1,2]. Although hydrodynamic disorders play an important role in the pathogenesis, these forms of syringomyelia, several aspects of this disease are dif icult to explain only on the basis of hydrodynamics. Different theories of pathogenesis of syringomyelia were proposed such as “cranio-spinal pressure dissociation”, transmedullary passage of CSF, impaired venous drainage [2,3]. However, the precise pathogenesis of “hindbrain related” syringomyelia is unknown. There are some references of possible role antibodies in the development of syringomyelia. Researches in this ield are of great interest. It is undoubted, that experimental studies can be used in the explanation of the nature of this important group of neurological disorders, as we do not have possibilities to study directly the human central nervous system.


Introduction
Numerous experimental observation and clinical researches demonstrated that the majority of the forms of syringomyelia are found in combination with inherent or acquired hindbrain abnormalities [1,2]. Although hydrodynamic disorders play an important role in the pathogenesis, these forms of syringomyelia, several aspects of this disease are dif icult to explain only on the basis of hydrodynamics. Different theories of pathogenesis of syringomyelia were proposed such as "cranio-spinal pressure dissociation", transmedullary passage of CSF, impaired venous drainage [2,3]. However, the precise pathogenesis of "hindbrain related" syringomyelia is unknown. There are some references of possible role antibodies in the development of syringomyelia. Researches in this ield are of great interest. It is undoubted, that experimental studies can be used in the explanation of the nature of this important group of neurological disorders, as we do not have possibilities to study directly the human central nervous system.
The aim of the present experimental study was to determine a possible role of lymphatic drainage of the CSF and antigens stimulation of regional lymph nodes in induction of demyelinating lesions for "hindbrain related" syringomyelia.

Matherial and Methods
A study was approved by the Ethical Committee of Scienti ic Center of Reconstructive and Restorative Surgery. In this study we used 26 New Zeland white rabbits, each weighing 3500 ± 350 g, and the animals were allocated into two groups. Prior the operation all animals endured intramuscular cephazolin sodium injection (20 mg/kg). All procedures were performed while the rabbits were under pentobarbital anesthesia.
Experimental syringomyelia was induced in 20 anesthetized rabbits by injecting 0.5 ml of 25% kaolin suspension into the cisterna magna. In addition, 6 rabbits with puncture and injection sterile saline NaCl were used as a control.
The animals were sacri iced 1, 2, 4 and 6 months (5 animals per period) after the kaolin injection. Four hydrocephalus rabbits (two animals with 4 and two with 6 months of experiment) were sacri iced in 17 hours after the puncture of lateral ventricle with injection of solution of colloidal gold labeled human albumin (0.5 mg HAuC14 in 0.2 ml sterile saline). For sacri icing the animals were anesthetized by intravenous pentobarbital (50 mg/kg). Their descending aorta was clamped, and they were perfused via transcardiac path with 4% paraformaldehyde in 0.12 M phosphate buffer and 0.02 mM calcium chloride at 37 °C. After ventricular injection of gold labeling albumin retropharyngeal lymph nodes of the animals were examined by electron microscopy. The sections of the brain and spinal cord were stained with hematoxylin and eosin by Nissle and Marchi methods and with immunogold technique. The preparations were examined under a light microscope with magni ication ×40, 100, 200 and 400. Meningeal ibrosis was examined according to the following scheme designed by He et al. The ibroblast cell density was calculated in each ield by the median ibroblast cell count.

Statistical Analysis
Data analysis was performed using SPSS for Windows, version 11.5 [SPSS Inc., Chicago, IL, USA]. The Shapiro-Wilk test was used to determine if the distributions of continuous variables were normally distributed. The Bartlett test was used to evaluate the homogeneity of the variances. Continuous and ordinal variables were shown as medians (min-max). A nonparametric Kruskal-Wallis test was used to determine the statistical signi icance of the pachymeningeal ibrosis extension, in lammation, intramedullary cyst formation and dilatation of the ventricular system among groups. The differences in the median values among the groups were compared using a Mann-Whitney test. We used the Likelihood Ratio test to determine whether the differences in nominal data were statistically signi icant. When the p values from the Likelihood Ratio test statistics were statistically signi icant, we employed Wilcoxon's exact test to determine which group differed from which other groups. A p value less than 0.05 was considered statistically signi icant.

Results
The use of the experimental model of kaolin induced syringomyelia showed in the rabbits the following disturbances in CNS. Intramedullary cavities similar to syringomyelia appeared in the cervical, thoracic and lumbar cord ( igure 1). During the early stage of the experiment (2 -4 months) in lammatory in iltrates were found around vessels in the meninges, and rarely ind in white matter. Sometimes in lammations resulted in honeycombs-like destructions of nervous tissue ( igure 2).
In all the animals there was found hydrocephalus but with some variation in the degree of dilatation of the lateral ventricles and of the central lumen of the olfactory bulbs ( igure 3-7).
There was signi icant concordance on all parameters between 2 observers (k coef icient 0.684, 0.712 and 0.702 for pachymeningial ibrosis, intramedullary cyst formation and dilatation of the ventricular system). All of these values were statistically signi icant (p<0.001).
Fibroblast cell density and meningeal ibrosis were to be less in the control group than in the experimental group (p=0.003, and p=0.005, respectively); these differences were statistically signi icant also (table 1).
After 6 months the experiment of cyst formation, demyelinated plagues in   combination with swollen astrocytes were observed. Ultrastructural examination of retropharyngeal lymph nodes of the animals after intraventricular injection of gold labeled albumin revealed gold particles in lysosomas of reticular cells in lateral sinus.

Discussion
It is generally accepted that chronic pachymeningitis of the hindbrain as well as hindbrain anomaly leads to CSF circulation disorders on the level of fourth ventricle    outlets. It is an initial mechanisms of dilatation of the ventricular system and cavity formation within the spinal cord. Although hydrodynamic disorders play an important role in the pathogenesis of hydrocephalus and "hindbrain related" syringomyelia, several aspects of disease are dif icult to explain only on the basis of hydrodynamics [1,2]. Moreover, indirect evidences show that immune reactions against CNS antigens may play an additional role in the demyelination for "hindbrain related" syringomyelia. However, we cannot explain whether Ig synthesis take place in syringomyelia patients. Blood-brain barrier (BBB) damage is probably an important factor in the problem of determining whether Ig was synthesized in CNS or entered the brain as a result of BBB changes.
Obviously, that the immunological disorders in syringomyelia are of secondary character. Therefore, the following question arises: Why does selective sensibilization against CNS tissue occur. According to the presented data, it is clear if at some moment of time a mitogenic dose of neuroantigen reaches draining lymph nodes, then the proliferation of speci ic neuroantigen-sensitized cells occur. This mechanisms allows auto reactive clones of lymphoid cells to act directly against CNS antigens. Lymphatic drainage of the CSF has been well documented in a number of mammalian species [4,5]. Then, by highly directionalised pathways, CSF drains to the olfactory bulbs and through arachnoid channels to connect with lymphatic vessels in the nasal mucosa and deep cervical lymph nodes. Our observation on albumin-gold complex reaching to retropharyngeal lymph nodes after ventricular injection of tracer showed that water hammer effect and internal destruction of the spinal cord may lead to continuous antigen stimulation of regional lymph nodes. Anatomical pathways for lymphatic drainage of interstitial luid, analogous to those in the animals, are present in the human brain [6,7]. It testi ies that lymphatic system of head and neck take part in CSF resorbtion. Therefore, it is possible that in humans the continuous antigen stimulation of regional lymph nodes may lead to the induction of chronic demyelination as it happens in the experimental animals. Cervical lymphatic lymph nodes play signi icant roles in the induction of immunological tolerance and of adaptive immunological responses. The identi ication and characterization of antigens involved in the pathogenesis of